Aspergillus fumigatus is the most prevalent airborne fungal pathogen, causing severe and usually fatal invasive infections in immunocompromised hosts. Success of anti-fungal therapy relies heavily on accuracy of diagnosis, but rapid and reliable detection of A. fumigatus remains difficult owing to the non-specific clinical symptoms and lack of distinct molecular targets. This proposal aims to utilize thermostable proteases secreted by A. fumigatus into the host environment as a target for the development of a novel fluorescence-based diagnostic assay for invasive aspergillosis. The applicant hypothesizes that fungal proteolytic activity can be detected in serum and BAL samples during invasive aspergillosis using a panel of Internally Quenched Fluorogenic Probes (IQFPs). Aim 1 of the R21 phase will use a commercially available library of 360 IQFPs to optimize assay conditions for maximal detection of fungal proteolytic activity secreted in vitro. These conditions will then be used to test the feasibility of the assay for the detection of A. fumigatus in serum and BAL samples from animal models of infection (Source: IAAM contract). In Aim 2, a panel of eight IQFPs with most promising diagnostic properties will be selected for targeted optimization in order to maximize sensitivity and selectivity. The R33 phase we will then evaluate the efficacy of this IQFP-based diagnostic platform for the detection of A. fumigatus proteases in clinical samples from human patients (Source: AsTeC contract). In addition, a comprehensive combinatorial library of 640,000 IQFPs will be assembled in order to expand the diagnostic applications to include the monitoring of antifungal therapy and the detection of different pathogenic moulds.